Compound B as an angiogenic agent in combination with human growth factors

ABSTRACT

The present invention refers to the use of Compound B as angiogenic agent in combination with human growth factors.

FIELD OF THE INVENTION

The present invention refers to the use of Component B as angiogenicagent in combination with human-growth factors.

STATE OF THE ART

Component B (hereinafter indicated as CB) is a 81 amino acid proteinoriginally isolated from human urine. The human gene expressing theprotein has been cloned and expressed in CHO cells as recombinant humanComponent B, the protein has a molecular weight of about 8.9 kD and wasthoroughly described in WO 94/14259 to which reference is made also forthe methods of preparation and its amino acid sequence.

In WO97/39765 the use of CB as cicatrizant was described.

It is also known that growth factors, as for example basic fibroblastgrowth factor (bFGF) or vascular endothelial growth factor (VEGF) haveangiogenic activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows the implant of a pellet loaded with both active compounds(1A) or of two pellets each loaded with one active compound (1B) in therabbit cornea.

FIG. 2: Shows the effect of bFGF and CB on angiogenesis in the rabbitcornea.

FIG. 3: Shows the effect of heat-inactivation on the angiogenic activityof CB.

FIG. 4: Shows the synergistic effect of CB on bFGF-induced angiogenesis.

FIG. 5: Shows the synergistic effect of CB on the angiogenic activity ofbFGF.

FIG. 6: Represents a drawing of a typical histological section of rabbitcornea illustrating the main structures observable in following FIGS.7-11.

FIG. 7: section of rabbit cornea (×200) with a corneal pocket containing100 ng of bFGF sampled at 6 days post-surgery wherein the arrows showneoformed vessels. (Ep=epithelium).

FIG. 8: section of rabbit cornea (×100) with a corneal pocket containing500 ng of CB sampled at 2 days post-surgery wherein the arrows showneoformed vessels.

FIG. 9: section of rabbit cornea (×200) with a corneal pocket containing500 ng of CB sampled at 6 days post-surgery wherein the arrows showneoformed vessels.

FIG. 10: Section of rabbit cornea (×100) with a corneal pocketcontaining 4 μg of CB sampled at 15 days post-surgery wherein the arrowshow neoformed vessels.

FIG. 11: Section of rabbit cornea (×100) with a corneal pocketcontaining 500 ng of bFGF sampled at 15 days post-surgery wherein thearrow show neoformed vessels.

FIG. 12: mean and 95.0% Tukey HSD Intervals

DETAILED DESCRIPTION OF THE INVENTION

It was now surprisingly found that the simultaneous presence of CB and agrowth factor increases the angiogenic response elicited by eitherindividual agent, in other words CB is capable of synergizing with agrowth factor in promoting neovascular growth in the tissues probablyfacilitating some early events required to mobilise endothelial cellsfrom capillaries.

Therefore, the main object of the present invention is the use of CB incombination with a growth factor for the manufacture of pharmaceuticalcompositions useful for the treatment of wounds, ulcers and othertraumatic lesions to any of the tissues in the body.

Another object of the invention are pharmaceutical compositions preparedas described above.

A further object of this invention is a method of treatment of wounds,ulcers and other traumatic lesions to any of the tissues in the body,comprising administering an effective amount of CB and of a growthfactor, possibly together with a pharmaceutically acceptable excipient.

The administration of the active ingredient may be oral, intravenous,intramuscular, subcutaneous or topical. Other routes of administration,which may establish the desired blood levels of respective active agentsare comprised by the present invention.

The administration of the two active compounds can be performed by asingle pharmaceutical preparation containing both of them or,preferably, by two pharmaceutical preparation each containing separatelyone of the two ingredients. Preferred growth factor to be used incombination with CB according to the invention are the basic fibroblastgrowth factor (bFGF) or the vascular endothelial growth factor (VEGF).

Angiogenesis was studied in the cornea of albino rabbits since this isan avascular and transparent tissue where inflammatory reactions andgrowing capillaries can be easily monitored and changes quantified bystereomicroscopic examination (Ziche et al., 1982). This method allowsthe monitoring over an exended period of time of vessel growth by directand non traumatic observation of the process. Moreover in the sameanimal the quantification of the effect can be compared to that of aknown agent.

The investigation of the role of Component B (CB) in in vivoangiogenesis studied in the rabbit cornea assay was performed by:

-   -   a) testing the ability of the molecule to produce vessel growth        when placed into the avascular corneal stroma;    -   b) testing the ability of the molecule to favour or repress        neovascularization elicited by the angiogenesis factor basic        fibroblast growth factor (bFGF) or vascular endotelial growth        factor (VEGF).        Methods        Protocol for Slow-Release Preparation of Test Compounds

Growth factors or peptides-were prepared as slow-release pellets.

Slow-release pellets (1×1×0.5 mm) were prepared in sterile conditionsincorporating the test substances into a casting solution of aethinyl-vinyl copolymer (Elvax40, Dupont, Wilmington, Del.), in 10%methylene chloride (10 μl/droplet) (Langer and Folkman, 1976; Ziche etal. 1982).

Surgical Procedure

The angiogenic activity was assayed in vivo using the rabbit corneaassay. In the lower half of New Zealand white rabbit eye (Charles River,Calco, Lecco, Italy), anaesthetised by sodium pentothal (30 mg/kg), amicro pocket (1.5×3 mm) was surgically produced using a pliable irisspatula 1.5 mm wide.

The pellets were implanted in the micropockets located into thetransparent avascular corneal stroma.

Quantification of Corneal Angiogenesis

Subsequent daily observations of the implants were made with a slit lampstereomicroscope without anaesthesia. An angiogenic response was scoredpositive when budding of vessels from the limbal plexus occurred after3-4 days and capillaries progressed to reach the implanted pelletaccording to the scheme previously reported (Ziche et al. 1989).Angiogenic activity is expressed as the number of implants exhibitingneovascularization over the total implants studied. Potency is scored bythe number of newly formed vessels and by their growth rate. Data areexpressed as angiogenesis score, calculated as vessel density×distancefrom limbus in mm. A density value of 1 corresponded to 0 to 25 vesselsper cornea, 2 from 25 to 50, 3 from 50 to 75, 4 from 75 to 100 and 5 formore than 100 vessels (Ziche et al., 1994).

Experimental Design

The effect of Component B was tested following two procedures:

-   -   A) Three different concentrations of the molecule were tested in        the cornea of at least 4 distinct rabbits per each dose, to        define the potential angiogenic activity of the compound. The        effect of Component B was compared with that elicited by the        growth factor bFGF at 50 and 100 nag/pellet. In this        experimental protocol rabbits were monitored for 3 weeks.    -   B) To evaluate a potential role of Component B in modulating        angiogenesis the effect of this agent was tested in the presence        of a defined angiogenesis factor, i.e. bFGF. To this aim two        adjacent pockets were surgically produced in the same cornea,        one bearing the angiogenic trigger and the other Component B.        Experiments were also performed testing both substances        incorporated into the same pellet (FIG. 1).

This last experimental protocol was specifically set up by our group todefine:

-   -   1) the effect of the agent as a “costimulator” of the        angiogenesis elicited by bFGF; or 2) the ability of the agent to        inhibit angiogenesis elicited by the. growth factor (Ziche et        al, 1992 and 1994).

In this experimental protocol the rabbits were monitored for 4-5 weeks.The same protocol was used to tests the effect of CB as “costimulator”of the angiogenesis induced by VEGF.

Histological Analysis

Rabbit corneas with corneal pockets containing CB and/or bFGF weresampled at 2, 6, 15 days post surgery, and fixed in formalin afterremoving the pellets. Routine histopathological processing wasperformed; sections 5 μm thick were cut next to where each pellet wasplaced; sections were stained with hematoxylineosin. At least 40sections were examined per each cornea.

Statistical Analysis

Results are expressed as means for (n) implants. Angiogenic score datacontained both positive and negative results. Multiple comparisons wereperformed by one-way ANOVA and individual differences were tested byFisher's test after the demonstration of significant intergroupdifferences by ANOVA. A P value <0.05 was taken as significant (see alsoAppendix for further statistical evaluation).

Results

a) Angiogenic Activity of CB

The angiogenic activity of CB was tested after incorporating increasingconcentrations of the compound in slow release-pellets of thepolymer-Elvax40. Solubilization and incorporation of the compound intothe polymer pellets did not cause any specific problem. The doses testedwere: 0.2, 0.5, 2 and 4 μg/pellet. The effect of CB was compared to thatproduced by basic fibroblast growth factor (bFGF).

CB elicited a dose-dependent angiogenic effect whose potency appeared tobe weaker than the one elicited by bFGF. In FIG. 2A data are reported,on the angiogenic activity of bFGF obtained from previous experimentsand from the experiments run in parallel with CB. In FIG. 2B data fromdaily observation of rabbit corneas implanted with CB-containing pelletsare reported as angiogenic score. The highest angiogenic score obtainedwith CB averaged around 3-3.5 (2-4 μg/pellet) (P<0.05 vs vehicle pelletsalone) vs 7-8 produced by bFGF (0.2 μg/pellet)(P<0.05 vs vehicle pelletsalone). CB was not angiogenic at the concentration of 0.2 μg/pellet. Asshown in Table 1, 0.5 μg/pellet CB induced a positive angiogenicresponse in 1 implant out of 5 performed. Two and 4 μg/pellet were themost effective doses. These doses induced a similar angiogenic activityand produced 2 positive implants out of 5 performed.

CB was devoid of any macroscopic inflammatory activity as revealed bythe persistence of corneal transparency all through the experiments atany concentration tested.

To assess the specificity of CB angiogenic effect, the compound washeat-inactivated (h.i.) by boiling it for 20 min. The dose of 2 μg wasthen tested. Following heat inactivation CB completely lost angiogenicactivity (FIG. 3)(P<0.05 5 vs CB 2 μg).

b) Effect of CB on the Angiogenesis Induced by bFGF

To evaluate the potential role of CB in modulating the effect of a knownangiogenic effector, experiments were performed testing suboptimalconcentrations of both substances (500 ng of CB and 100 ng of bFGF)co-released into the corneal stroma. Experiments were performed testingboth substances incorporated into the, same pellet (FIG. 1A).Furthermore the compounds were tested at the same-concentration as abovebut released into the stroma separately in 2 independent pellets (FIG.1B).

The simultaneous presence of CB and bFGF into the cornea increased theangiogenic response elicited by either individual agent (FIGS. 4A and B,Table 2).

Angiogenesis occurred earlier and progressed more rapidly producing asignificant increment of the number of newly formed vessels (P<0.05 vsCB and bFGF alone). This effect was apparent in both experimentalconditions.

However, when CB and bFGF were released independently by 2 separatepellets the effect was higher. Capillaries grew toward bFGF rather thanCB suggesting that CB contributed to potentiate bFGF activity. After 7days, neovascular growth started to regress.

Additional experiments were performed with increasing concentrations ofCB (0.2, 0.5 and 2 μg/pellet) on the angiogenesis elicited by a constantconcentration of bFGF (100 ng). A synergism between the two moleculescould be observed (FIG. 5). Interestingly, the most effective conditionof synergism between CB and bFGF was observed with 200 ng CB (P<0.05 vsCB and bFGF alone) tested in two separate pellets.

c) Effect of CB on the Angiogenesis Induced by VEGF

In Table 3 the synergistic effect of Component B on VEGF-inducedangiogenesis are reported.

The synergism between CB and VEGF was evaluated with the factors testedin two separate pellets. The results obtained at day 10 are reported inTable 3. The data are expressed as the number of implants exhibitingneovascularization with an angiogenesis score equal or over to 6, overthe total implants performed.

Further statistical analysis was performed in order to confirm possiblepositive interactions between the test compounds using a moreconservative analysis (see FIG. 11 in Appendix).

The main factors (“test compounds” and “angiogenic score” over time)were analysed according to the Multifactor Analysis. The results showedthat statistically significant differences (p<0.0001) are present amongthe test compounds over the time.

As to the interaction among the test compounds the results of theTukey's test allow the following considerations (see FIG. 12 below):CB 500 ng+bFGF 100 ng (1 pellet)

Both compounds individually are not statistically different fromcontrols. The combination of the two compounds gave a response which isstatistically different from either the controls and the single drugs.The response is around the expected additive effect.CB 200 ng+bFGF 100 ng (2 pellets)

Both compounds individually are not statistically different fromcontrols. The combination of the two compounds gave a response which isstatistically different from either the controls and the single drugs.In addition, it should be noted that the response of the combinedtreatments clearly exceeds the expected additive effect. The above seemsto confirm the presence of a synergistic effect between the two drugs.CB 500 ng+bFGF 100 ng (2 pellets)

Both compounds are not statistically different from controls. Thecombination of the two compounds gave a response which is statisticallydifferent either from the controls and the single drugs. The response isaround the expected additive effect. In addition, no differences werefound comparing CB 500 ng+bFGF 100 ng (1 pellet) vs CB 500 ng+bFGF 10 ng(2 pellet).CB 2 μg+bFGF 100 ng (2 pellets)

Both compounds are not statistically different from controls. Thecombination of the two compounds gave a response which is statisticallydifferent from controls but not from the single compounds. The responseis around the expected additive effect.

Histological Analysis

The effect of CB was examined at the maximal effective concentration (4μg) and at a suboptimal concentration (500 ng) in the presence and inthe absence of bFGF (100 ng). No difference in the extent of cellularinfiltrate was apparent between CB and bFGF implants in any combination(FIGS. 7,8,9). Within 2 days from the implants a leukocyte infiltratesurrounded a dense network of newly formed capillaries in proximity ofthe limbal region at the epithelial side of the cornea (FIG. 10). At day6 a consistent reduction in the extent of the leukocyte infiltrate wasapparent while capillary vessels appeared increased in number andcaliber in response to either molecule (FIG. 11). At day 15 the extentof the leukocyte infiltrate was negligible while capillaries appearedmorphologically unmodified.

Conclusions

Component B possesses angiogenic activity which is apparent in theconcentration range of micrograms and which is lost by heatinactivation. Most of the angiogenesis factors are angiogenic atconcentrations 20-40 fold lower. Together with the high concentrationrequired to elicit angiogenesis, 2 aspects appear relevant in CB effect:

-   -   1) the ability to elicit budding of capillaries within the first        3-4 days from the implant, mimicking the secreted angiogenesis        factor VEGF rather than the matrix linked angiogenesis factor        bFGF;    -   2) the flattening over time of the efficiency of neovascular        growth, leading to only 30-40% of the implant tested to be fully        vascularized after 10-14 days.

Our results indicate that CB induces angiogenesis in vivo and has theability to synergize with bFGF in promoting neovascular growth in therabbit cornea.

These considerations together with the characteristics of thepotentiation of the angiogenic response in the presence of bFGF, suggestthat CB requires the presence of additional growth factors to fullyexpress its angiogenic potential in vivo.

Histological examination of corneal sections sampled at various timeintervals was performed to assess whether the angiogenesis processelicited by CB involved inflammatory cell infiltrate. The effect of CBwas compared to that produced by the corneal implant of bFGF. At routinehistological examination we did not find major differences in the extentand in the type of leukocyte infiltrate in corneas receiving CB, bFGF orthe combination of the two. Thus from our results we can conclude thatthe corneal vascularization induced by CB does not appear to be mediatedby gross inflammatory reaction products since no sign of corneal opacitywas apparent.

The characteristic of the angiogenic response elicited in the avascularcornea by CB suggests that CB might facilitate some of the early eventsrequired to mobilise endothelial cells from capillaries. Once thisprocess is started and endothelial cells are “loosened” from the tightboundary to the extracellular matrix, bFGF expresses its mitogeniceffect with more effciency. TABLE 1 Effect of Component B onangiogenesis in the rabbit cornea Positive Positive CB implants/totalbFGF implants/total (μg/pellet) performed (ng/pellet) performed 0 0/6 00/6 0.2 0/5 50 1/6 0.5 1/5 100 2/6 2 2/5 200 5/6 4 2/5Data are expressed as positive implants exhibiting neovascularizationover the total implants performed. The results obtained at day 7 arereported.An angiogenic response was scored positive when budding of vessels fromthe limbal plexus occurred after 3-4 days and capillaries progressed toreach the pellet containing the angiogenic factors.

TABLE 2 Synergistic effect of Component B on bFGF-induced angiogenesisPositive implants/ Test compounds total performed CB 500 ng 1/5 bFGF 100ng 1/5 CB 500 ng + bFGF 100 ng (one pellet) 3/6 CB 500 ng + bFGF 100 ng(two pellets) 4/5Data are expressed as positive implants exhibiting neovascularizationover the total implants performed. The results obtained at day 7 arereported.An angiogenic response was scored positive when budding of vessels fromthe limbal plexus occurred after 3-4 days and capillaries progressed toreach the pellet containing the angiogenic factors.

TABLE 3 Synergistic effect of Component B on VEGF-induces angiogenesisPositive implants/ total performed CB 200 ng 0/5 CB 500 ng 1/5 VEGF 100ng 1/4 CB 200 ng + VEGF 100 ng 2/4 CB 400 ng + VEGF 100 ng 4/4

1. A method of promoting angiogenesis in a patient in need thereof,comprising administering to the patient Component B and a human growthfactor, for a time sufficient and in amounts effective for the promotionof angiogenesis in the patient, wherein said Component B and said humangrowth factor are administered such that they will be simultaneouslypresent in the blood of the patient in amounts effective for thepromotion of angiogenesis in the patient.
 2. The method of claim 1,wherein the angiogenesis is in relation to the treatment of a wound,ulcer or other traumatic lesion to the tissues of the body of thepatient.
 3. The method of claim 1, wherein the Component B and the humangrowth factor are administered in a single composition.
 4. The method ofclaim 2, wherein the Component B and the human growth factor areadministered in a single composition.
 5. The method of claim 1, whereinthe Component B and the human growth factor are administered in separatecompositions.
 6. The method of claim 2, wherein the Component B and thehuman growth factor are administered in separate compositions.
 7. Themethod of claim 1, wherein the ratio of the amount of Component B to theamount of the human growth factor is selected to provide synergisticangiogenesis results.
 8. A method of treating a wound, ulcer or othertraumatic lesion in a patient in need thereof, comprising administeringto the patient Component B and a human growth factor, for a timesufficient and in an amount effective for the treatment of the wound,ulcer or other traumatic lesion in the patient, wherein said Component Band said human growth factor are administered such that they will besimultaneously present in the blood of the patient in amounts effectivefor the treatment of the wound, ulcer or other traumatic lesion in thepatient.
 9. The method of claim 8, wherein the Component B and the humangrowth factor are administered in separate administration doses.
 10. Themethod of claim 8, wherein the ratio of the amount of Component B to theamount of the human growth factor is selected to provide synergisticresults.
 11. A pharmaceutical composition for promoting angiogenesis,comprising Component B and a human growth factor, in combination with apharmaceutically acceptable carrier, wherein the ratio of the amount ofComponent B to the amount of human growth factor is selected to providesynergistic angiogenesis results when administered to a patient in needthereof.
 12. A pharmaceutical composition for treating a wound, ulcer orother traumatic lesion, comprising Component B and a human growthfactor, in combination with a pharmaceutically acceptable carrier,wherein the ratio of the amount of Component B to the amount of humangrowth factor is selected to provide synergistic results whenadministered to a patient in need thereof.
 13. A method for promotingangiogenesis in a patient in need thereof, comprising administering tothe patient an effective amount of Component B.
 14. The method of claim13, wherein the angiogenesis is in relation to the treatment of a wound,ulcer or other traumatic lesion to the tissues of the body of thepatient.